16SrDNA克隆文库方法分析两纯系鸡回肠及盲肠黏膜细菌的组成

Analysis of Composition of the Mucosa-associated Microbiota in Ileum and Cecum between Two Chicken Pure Lines by 16S rDNA Clone Library

以1日龄纯系的优质肉鸡A系(♂,A组)和惠阳胡须鸡(♂,B组)为试验动物,研究比较两纯系鸡28日龄回肠与盲肠黏膜菌群的组成及多样性。于第28日龄分别从2组中采集鸡2个肠段并提取黏膜细菌基因组DNA,应用16S rDNA基因序列技术,建立2个肠段黏膜细菌16S rDNA V3区的随机克隆文库。回肠文库分析发现,A组文库有86个序列共产生6个OTUs()perational taxonomic units),其已知序列主要属于肠球菌属、乳杆菌属、粪球菌属及梭菌属类序列,而B组文库有97个序列共产生2个OTUs,主要属于梭菌类序列;盲肠文库分析发现,A组文库有93个序列共产生44个OTUs,其优势菌群是未分类瘤胃球菌属(18.28%)、粪球菌属(13.98%)、拟杆菌属(10.75%)、Blautia(10.75%)及未分类毛螺菌属(7.53%);B组文库有97个序列共产生42个OTUs,其优势菌群是普拉梭杆菌属(17.53%)、粪球菌属(11.34%)、拟杆菌属(9.28%)及未分类瘤胃球菌属(8.25%);2组盲肠文库中其他菌属的数量存在较大差异,且未发现与大肠杆菌相关的序列。优质肉鸡A系和惠阳胡须鸡28日龄回肠与盲肠黏膜细菌的组成差异明显,尤其是瘤胃球菌属与产丁酸菌的组成比例,这种差异可能是宿主遗传的影响所致。

The experiment was conducted to study the composition and diversity of the mucosa-associated microbiota(MAM) in ileum and cecum between high quality chicken line A(pure line,group A) and Huiyang Beard chicken(pure line.group B) at 28 days old.Two chicken intestinal tracts were collected in group A and B,and mucosa-associated microbiota DNA were extracted at 28 days old,respectively.The random clone library of 16 S rDNA V3 region of the mucosa-associated microbiota(MAM) in ileum and cecum between group A and B was established by analyzing 16 S rDNA gene sequences.Analysis of the library in ileum showed that 6 operational taxonomic units(OTUs)(form 86 gene sequences or clones) were detected in the group A,as compared with 2 OTUs(form 97 clones) in the group B.Sequence being related to Enterococcus,Lactobacillus,Coprococcus and Clostridiaceae were found in the clone library of the MAM in ileum in group A.Clostridiaceae was only detected in group B.Analysis of the library in cecum shown that 44 OTUs(form 93 clones) were detected in the group A,as well as 42 OTUs(from 97clones) in the group B.Unclassified Ruminococcaceae(18.28%),Coprococcus(13.98%),Bacteroides(10.75%),Blautia(10.75%) and Unclassified Lachnospiraceae(7.53%) were predominant in the group A,and Faecalibacterium(17.53%),Coprococcus(11.34%),Bacteroides(9.28%) and Unclassified Ruminococcaceae(8.25%) were abundant in the group B.The amount of other species found in cecum in group A were significant different from that in group B.In addition,sequence known being related to Colibacillus were not detected in group A and B.These results indicate that the composition of the MAM in ileum and cecum at day 28 days old are different between the high quality chicken line A and Huiyang Beard chicken,especially the proportion of butyric acid bacteria and Ruminococcus,and the host genetic factors may plays key role in this difference.